Diploid human cells are believed to inherently exhibit limited life-span in vitro. Key evidence in support of the cellular senescence hypothesis was first reported by Hayflick and Moorhead about thirty years ago (Hayflick and Moorhead, 1961; Hayflick, 1979), who showed that diploid embryonic human lung fibroblasts in vitro could be cultivated for a short period and undergo only a limited, fixed number (50-60) of population doublings. It was further reported that the number of doublings that a cell population could survive was inversely related to the donor's age. Escape from senescence, which occurred mostly in cancer cells or experimentally transformed cells, was always accompanied by numerical and/or structural chromosomal alterations (Wolman, 1991); Fusenig et al. 1991). Immortality of cells has been induced by chemical carcinogens, oncogenes and viruses (Zeitlin et al. 1991, Chopra et al., 1991, Rhim et al. 1986, Stoner et al., Christian et al, 1987, Popescu and Dipaolo, 1990). All immortalized cell lines reported so far exhibit at least one transformed phenotype such as focus formation and/or anchorage independent growth (AIG) and certain relatively non-specific chromosomal aberrations. To date, there are no immortal epithelial cell lines which exhibit normal diploid chromosome complement.
Many investigators have utilized transformed epithelial cell culture models to investigate biochemical and molecular mechanisms of growth and differentiation, intercellular communications, toxicity and carcinogenicity of chemicals, oncogenes and cytokine expression, and activities of hormones. Results of these studies however, are severely flawed, because they are performed on cells exhibiting chromosomal aberrations involving undefined genetic alterations. Because of these chromosomal aberrations, the usefulness of transformed cells in research is greatly limited. For instance, if the cells have chromosomal alterations prior to a test treatment, it is difficult to determine specific genetic alterations associated with the treatment. For carcinogenesis studies, other investigators have used fibroblast cultures which usually can be propagated for longer duration than epithelial cells. However, the analogy between the two cell types (fibroblast and epithelial) is highly questionable because most cancers originate from cells of epithelial origin. More specifically, the lack of availability of diploid epithelial cells with an extended life-span has hindered cancer research in particular, and biomedical research in general.
In studies on propagation of epithelial cells in vitro, serum is frequently employed as a growth supplement. More recent studies have demonstrated that serum contains numerous undefined growth inhibitory and toxic factors. For example, TGF-.beta., a major component of serum, has been shown to inhibit the growth of a variety of cell types (Masui et al., 1986, Loo et al., 1987, Miyashita et al., 1989). Additionally, high concentrations of proteases and chelating agents are repeatedly used for serial passaging of cell cultures and may also cause irreparable damage to cells. Thus, it is not surprising that continuous culturing of cells in serum-containing medium and repeated treatments with proteases and chelating agents ultimately causes the death of normal cells.
U.S. Pat. No. 4,980,290 entitled "Human Uroepithelial Cell" describes cell lines derived from the human ureters and immortalized by SV40 virus; those cells show preneoplastic phenotypes and gross chromosomal aberrations and they are maintained in serum-supplemented medium. U.S. Pat. No. 5,026,637 entitled "Immortal Human Mammary Epithelial Cell Lines" are maintained in serum-supplemented medium and show numerous chromosomal aberrations including several marker chromosomes. Neither of the processes described, nor the cells themselves, constitute normal human diploid immortal epithelial cell lines.
Therefore, it is most desirable to provide normal human epithelial cell lines which maintain a normal diploid chromosomal complement, produce tissue-specific proteins, and that could be cultivated indefinitely. It would also be desirable to provide such cell lines which could be cultivated in serum-free medium. It would be further desirable to provide such cell lines without infection and/or transfection with a virus and/or exogenous DNA. Such cell lines would be most desirable to the studies on carcinogenesis, aging, growth and differentiation, and to determine sensitivity to suspected reactive agents on a long-term basis. The present invention meets these objectives and provides two epithelial cell lines, HPAM1 and HPAF1, derived from the normal human salivary parotid gland, which maintain a normal diploid chromosome complement, produce the tissue-specific proteins amylase and proline-rich protein (PRP), and are cultivated indefinitely in serum-free medium. The invention also describes the process for preparing epithelial cells in such a manner so as to obtain the immortal salivary parotid gland cell lines.